Abstract. Their central importance to the synthetic, secretory and metabolic functions of the liver renders hepatocytes particularly vulnerable to injury. Reinforcing hepatocyte resilience to damaging insults holds potential to block liver disease progression at early stages, independent of etiology. To that end, ecto-5?-nucleotidase (CD73) is a promising novel liver disease target, as it plays critical roles in multiple mouse liver injury models, and is dysregulated in livers of patients with chronic liver diseases of multiple etiologies. The classical function of CD73 is metabolism of extracellular adenosine monophosphate (AMP) to adenosine. We found that global CD73-/- mice are resistant to chemically-induced hepatocellular injury, an unexpected finding given that liver injury induces near-complete loss of cell surface CD73 AMPase activity in wild-type mice. This points to alternative functions, which is also supported by our identification of a novel, catalytically-inactive CD73 isoform (CD73S) in advanced human liver disease. We observe differences in apoptosis-related signaling in primary hepatocytes expressing the two different human CD73 isoforms. CD73 binds two bioactive lipids in vitro, suggesting that it may regulate lipid homeostasis. Our central hypothesis is that non-enzymatic and alternative isoform-related mechanisms of hepatocyte CD73 promote liver injury and disease progression. The hypothesis will be addressed via two specific aims: (i) Define the non-enzymatic and isoform-specific functions of CD73 in hepatocytes and (ii) Examine the in vivo and tissue-level mechanisms of hepatoprotection in the absence of CD73. Our approach for Aim 1 will couple high throughput ligand screening, high content imaging, and proteomics with detailed biochemical, molecular and cell-biological assessments to determine if CD73 is a lipid-sensing receptor or a regulator of ER stress, and to determine the specific function of the non-catalytic CD73S variant in hepatocyte resistance to stress. For Aim 2, we will examine liver-specific contributions of CD73 in vivo using two models of alcohol and high fat diet-induced injury and our newly-generated liver-specific CD73-/- mice. We will utilize recombinant enzyme in vivo and precision-cut liver slices to perform mechanistic rescue and loss-of-function studies. This work will lead to a thorough integration of CD73 function in hepatocytes with the potential to yield novel mechanistic approaches for hepatoprotection.